Molecular Phylogenetics and Evolution 62 (2012) 764–776
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Molecular Phylogenetics and Evolution
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Phylogenetic relationships in Myrceugenia (Myrtaceae) based on plastid and nuclear DNA sequences ⇑ José Murillo-A. a, , Eduardo Ruiz-P. a, Leslie R. Landrum b, Tod F. Stuessy c, Michael H.J. Barfuss c a Departamento de Botánica, Universidad de Concepción, Casilla 160-C, Concepción, Chile b Department of Plant Biology, Arizona State University, Tempe, AZ 85287-160, USA c Department of Systematic and Evolutionary Botany, University of Vienna, Rennweg 14, Vienna 1030, Austria article info abstract
Article history: Myrceugenia is a genus endemic to South America with a disjunct distribution: 12 species occurring Received 10 July 2011 mainly in central Chile and approximately 25 in southeastern Brazil. Relationships are reconstructed Revised 31 October 2011 within Myrceugenia from four plastid markers (partial trnK-matK, rpl32-trnL, trnQ-50rps16 and rpl16) Accepted 22 November 2011 and two ribosomal nuclear regions (ETS and ITS) using maximum parsimony and Bayesian analyses. Rela- Available online 3 December 2011 tionships inferred previously from morphological data are not completely consistent with those from molecular data. All molecular analyses support the hypothesis that Myrceugenia is monophyletic, except Keywords: for M. fernadeziana that falls outside the genus. Chilean species and Brazilian species form two separate Blepharocalyx lineages. Chilean species form three early diverging clades, whereas Brazilian species are a strongly sup- cpDNA ETS ported monophyletic group in a terminal position. Least average evolutionary divergence, low resolution, ITS short branches, and high species diversity found in the Brazilian clade suggest rapid radiation. Geograph- Luma ical distributions and phylogenetic reconstructions suggest that extant Myrceugenia species arose in Myrteae northern Chile followed by colonization southward and finally to the Juan Fernández Islands and south- Myrceugenia eastern Brazil. South America Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Myrceugenia fernandeziana Hook. et Arn.) and five other genera into any group. He considered these genera to have arisen from the same Myrceugenia O. Berg is a South American genus consisting of ancestor as his six larger groups, but to have been less successful about 40 species, which exhibit a disjunct distribution: 26 species and therefore less species rich. Landrum (1981a) ‘‘tentatively’’ occur in southeastern Brazil and adjacent regions of Paraguay, Uru- accepted three subtribes with in the Myrteae based on embryo guay and northeastern Argentina, 12 are found in Chile and Andean structure and offered reason why these ‘‘may be true’’. He listed region of southwestern Argentina and two are endemic to the Juan seven anatomical, morphological and floral characteristics all found Fernández archipelago (Landrum, 1981a, 1981b). Myrceugenia in- in at least one genus in each subtribe and also found in the anom- cludes trees and shrubs with 4-merous flowers, usually persistent alous genus Luma. He speculated that the ancestral group that bracteoles, 2–4 locular ovaries, and few to several ovules per locule would link the three subtribes together would have these charac- (Landrum, 1981a). teristics and ‘‘provisionally’’ accepted these characteristics as plesi- Relationships with other members of tribe Myrteae are not clear. omorphies and the genera that have them as being similar to the Based on embryo structure Myrceugenia has been included in the ancestor of each subtribe. Thus, he hypothesized that Myrceugenia subtribe Myrciinae, but both inflorescence and floral characters is similar to the ancestor of the subtribe Myrciinae. Myrcianthes are similar to other Myrteae subtribes, Myrtinae and Eugeniinae. would hold a similar position in the Eugeniinae and Blepharocalyx In fact, many Myrceugenia species were originally included under in the Myrtinae. Luma was also hypothesized to belong to this group Eugenia L. and Myrtus L. (Landrum, 1981a). McVaugh (1968) divided of ancestral genera. Subsequent molecular work by Lucas et al. tribe Myrteae into six informal groups according to inflorescence, (2007) indicates that the Myrteae should be separated into seven flower, and seed characters, but he was unable to place Myrceuge- groups. One of these is the ‘‘Myrceugenia group’’ including Myrceu- nia, Luma A. Gray, Nothomyrcia Kausel (a monotypic genus for genia, Blepharocalyx and Luma. However this group has low support and only appears when nuclear and chloroplast markers are combined. ⇑ Corresponding author. Permanent address: Instituto de Ciencias Naturales, Species relationships within Myrceugenia have been investi- Universidad Nacional de Colombia, Apartado 7495, Bogotá, Colombia. Fax: +57 1 3165365. gated by Landrum (1981b) based on morphological characters. E-mail address: [email protected] (J. Murillo-A.). He used various methods and proposed various hypotheses and
1055-7903/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2011.11.021 J. Murillo-A. et al. / Molecular Phylogenetics and Evolution 62 (2012) 764–776 765 concluded that ‘‘there have very probably been three or more cases 2.3. Marker selection and primer design of transcontinental migration in Myrceugenia’’. Landrum (1981b) provisionally used two species from the Juan Fernández Islands Nine chloroplast markers were evaluated (matK gen and flank- as operational ancestors for his parsimony analysis, but stated that ing trnK intron, ndhA intron, rpl16 intron, intergenic spacer rpl32- the results should be considered undirected as the species were ac- trnL and flanking rpl32 gen, and the intergenic spacer psbA-trnH, cepted only ‘‘provisionally in order to use the program’’ and ‘‘ig- psbD-trnT, TrnL-trnF, trnQ-50rps16, following Shaw et al., 2007), nored in the final result.’’ For a final hypothesis based on but all of these showed low variability. However, we selected the numerical methods and general knowledge of the ecology of the four most variable regions (partial trnK-matK, rpl32-trnL, trnQ- species and suspected cases of hybridization, Landrum (1981b) 50rps16 and rpl16) for reconstructing phylogenetic relationships chose a point where branches to the two species of Juan Fernández among species of Myrceugenia. Internal transcribed spacer (ITS: Islands meet, based on their inflorescence structures. ITS1-5.8S-ITS2) of nuclear ribosomal genes 18S and 26S and the Analyses based on flavonoids (Ruiz et al., 1994) and genetic external transcribed spacer 18S-26S rDNA (ETS) were also divergences (Ruiz et al., 2004) have confirmed that the Juan included. Primers used for each region are listed in Table 2. Two Fernández species evolved in different lineages. These analyses, internal primers for region trnQ-50rps16 were designed. however, did not support the relationship between M. fernan- deziana and Brazilian species, but instead this species and 2.4. Amplification, sequencing and alignment Myrceugenia schultzei Johow were more related to other Chilean species. ITS sequences were amplified mainly in a volume of 25 lL con- In recent years great advances have been made in molecular taining 2.5 units of Taq polymerase (Paq5000 DNA polymerase, systematics (Felsenstein, 2004). This study of the molecular phy- Stratagene Inc.), 2.5 lL 10X PCR buffer (Stratagene Inc.), 0.2 mL of logeny of Myrceugenia was undertaken with the expectation that each dNTP (25 mM), 0.5 lL of each primer (4 lM). All other se- these methods, when applied to Myrceugenia, would result in a quences were amplified using 18 lL 1.1X ReddyMix PCR Master phylogenetic hypothesis with greater support than could be Mix (Thermo Fisher Scientific Inc, ABGene, UK), 0.4 lL of each pri- derived from morphological data. The present study is directed mer (20 lM), 0.6 lL 0.4% of bovine serum albumin (BSA, MBI-Fer- toward answering the following questions: What are the phyloge- mentas, St. Leon-Rot, Germany), and 1–2 lL template DNA. For netic relationships among Myrceugenia species? Are the species of reducing secondary structure problems 0.5 lL of dimethyl sulfox- Myrceugenia in Brazil a lineage completely distinct from those of ide (DMSO) were added to all nuclear markers amplifications. Chile or did they have a common evolutionary history? What is PCR conditions for ITS were according to the Stratagene polymer- the relationship between the endemic Juan Fernández species ase manufacturer’s recommendations. An initial DNA denaturation and those of the continent? at 95 °C for 2 min was followed by 30 cycles 95 °C for 20 s, 48–52 °C for 20 s, and 72 °C for 20 s, then a final extension at 72 °C for 5 min. PCR conditions for ETS were according to Lucas 2. Materials and methods et al. (2007). An initial denaturation at 94 °C for 5 min was fol- lowed by 30 cycles 94 °C for 1 min, 50 °C for 1 min, and 72 °C for 2.1. Taxon sampling 1 min, then a final extension at 72 °C for 5 min. PCR conditions for chloroplast regions were according to Shaw et al. (2007) with A total of fifty taxa were analyzed, with two individuals being some modifications. A first template denaturation at 80 °C for sampled in Blepharocalyx salicifolius (Kunth) O. Berg and Myrceuge- 5 min was followed by 35 cycles 94 °C for 30 s, 50–53 °C for 30 s, nia ovata (Hook. and Arn.) var. regnelliana (O. Berg) Landrum. All and a ramp of 0.3 °C/s to 65 °C, then 65 °C for 3 min, finally an species from Chile (12 species, including two varieties) and many extension at 65 °C for 8 min. from Brazil (25 species including nine varieties) were investigated. All amplicons were run on a 1% agarose gel to confirm amplifi- Eight species were included as outgroups: Luma apiculata (DC.) cation of PCR products. ITS amplified products were purified with Burret, Luma chequen (A. Gray), Blepharocalyx cruckshanksii (Hook. QIAquick PCR purification kits (Qiagen) according to manufac- and Arn.) Nied., B. salicifolius, all of these belonging to the ‘‘Group turer’s recommendations. All other PCR products were purified Myrceugenia’’ (Lucas et al., 2007), Myrcianthes cisplatensis (Cam- using 0.5 lL Exonuclease I and 1 lL Thermosensitive alkaline phos- bess.) O. Berg, Ugni molinae Turcz, Ugni selkirkii (Hook. and Arn.) phatase, FastAP (Fermentas) by incubation at 37 °C for 45 min, and O. Berg, and Myrtus communis L., the last one having been recog- later at 80 °C for 15 min. Most amplified ITS products were sent for nized as the sister group of tribe Myrteae (Lucas et al., 2007). All sequencing either to Macrogen (Korea) or to the Molecular Lab at DNA sequences analyzed were obtained during this study. Table the University of Santiago de Chile. All others sequences were 1 gives voucher specimens, herbarium, country of origin, and sequenced in the Molecular Laboratory of the Department of Sys- GenBank accession numbers. tematic and Evolutionary Botany in the Biodiversity Center of the University of Vienna. For cycle sequencing a 10 lL reaction volume including 1 lL of the primer (3.2 lM), 0.5 lL of BigDye Terminator 2.2. DNA extraction v3.1 Ready Reaction mix (Applied Biosystems, Austria), 1.75 lLof sequencing buffer, 4 lL of amplified purified product, and 2.75 lL
Total DNA genomic was extracted mainly from herbarium spec- of ddH2O was used. Cycle sequencing parameters consisted of an imens, in some cases from silica-gel dried or fresh leaves, using a initial 96 °C for 1 min, followed by 35 cycles 96 °C for 10 s, 50 °C modified CTAB (Cetyl trimethyl ammonium bromide) method for 5 s, and 60 °C for 4 min, finally 60 °C for 5 min. Electrophoresis (Doyle and Doyle, 1987). Ground material was previously treated was performed on a 3730 DNA analyzer (Applied Biosystems, ABI). several times with sorbitol, followed by incubation at 65–70 °C All sequences were analyzed and edited with Lasergene Seqman for 30 min with CTAB buffer and Sarkosyl. The precipitated mate- Pro 7.1.1 (DNASTAR) to obtain consensus sequences from both for- rial was left overnight at 20 °C and centrifuged at 14,000 rpm ward and reverse DNA strands. It was not possible to sequence for 30 min, then followed by two washes with 70% ethanol. Total either Myrceugenia bracteosa (DC.) D. Legrand and Kausel, Myrceu- DNA was resuspended in 30–50 lL of 1% TE buffer. Some samples genia hoehnei (Burret) D. Legrand and Kausel, Myrceugenia pilotan- were extracted using DNeasy extraction kit (Qiagen) according to tha (Kiaersk.) Landrum var. nothorufa (D. Legrand) Landrum, the manufacturer’s instructions. Myrceugenia scutellata D. Legrand, Myrceugenia venosa D. Legrand 766 J. Murillo-A. et al. / Molecular Phylogenetics and Evolution 62 (2012) 764–776
Table 1 List of taxa, voucher, country of origin, and GenBank accession numbers for plastid and nuclear sequences in species of Myrceugenia and outgroups.
Taxa Voucher (Herbarium) Origen trnQ-50rps16 rpl32-trnL rpl16 trnK-matK ETS ITS Blepharocalyx kruckshankii J. Murillo 4219 (CONC) Chile JN661105 JN661055 JN660956 JN661006 JN660857 JN660907 Blepharocalyx salicifolius L. Landrum 11232 (ASU) Argentina JN661134 JN661084 JN660985 JN661035 JN660886 JN660936 Blepharocalyx salicifolius M. Negritto 927 (CONC) Argentina JN661133 JN661083 JN660984 JN661034 JN660885 JN660935 Luma apiculata J. Murillo 4205 (CONC) Chile JN661108 JN661058 JN660959 JN661009 JN660860 JN660910 Luma chequen L. Landrum 7873 (CONC) Chile JN661109 JN661059 JN660960 JN661010 JN660861 JN660911 M. alpigena E. Lucas 167 (K) Brazil JN661090 JN661040 JN660941 JN660991 JN660842 JN660892 M. alpigena var. fuligenea G. Hatschbach 59697 (ASU) Brazil JN661089 JN661039 JN660940 JN660990 JN660841 JN660891 M. alpigena var longifolia R. Harley 26218 (ASU) Brazil JN661091 JN661041 JN660942 JN660992 JN660843 JN660893 M. brevipedicellata L. Landrum 2830 (ASU) Brazil JN661092 JN661042 JN660943 JN660993 JN660844 JN660894 M. campestris R. Kummrow 2940 (ASU) Brazil JN661093 JN661043 JN660944 JN660994 JN660845 JN660895 M. chrysocarpa L. Landrum 8166 (CONC) Chile JN661094 JN661044 JN660945 JN660995 JN660846 JN660896 M. colchaguensis L. Landrum 8033 (CONC) Chile JN661095 JN661045 JN660946 JN660996 JN660847 JN660897 M. correifolia S. Teillier 5360 (CONC) Chile JN661099 JN661049 JN660950 JN661000 JN660851 JN660901 M. cucullata R. Wasum 105 (ASU) Brazil JN661096 JN661046 JN660947 JN660997 JN660848 JN660898 M. euosma L. Soares 715 (ASU) Brazil JN661097 JN661047 JN660948 JN660998 JN660849 JN660899 M. exsucca J. Murillo 4217 (CONC) Chile JN661098 JN661048 JN660949 JN660999 JN660850 JN660900 M. fernandeziana T. Stuessy 15283 (CONC) Juan Fernández JN661101 JN661051 JN660952 JN661002 JN660853 JN660903 M. franciscensis P. Miyagi 357 (ASU) Brazil JN661100 JN661050 JN660951 JN661001 JN660852 JN660902 M. gerttii E. Barbosa 948 (ASU) Brazil JN661102 JN661052 JN660953 JN661003 JN660854 JN660904 M. glausecens L. Landrum 11231 (ASU) Brazil JN661103 JN661053 JN660954 JN661004 JN660855 JN660905 M. kleinii I. Cordeiro 734 (ASU) Brazil JN661104 JN661054 JN660955 JN661005 JN660856 JN660906 M. lanceolata M. Mihoc 6220 (CONC) Brazil JN661106 JN661056 JN660957 JN661007 JN660858 JN660908 M. leptospermoides J. Murillo 4214 (CONC) Chile JN661107 JN661057 JN660958 JN661008 JN660859 JN660909 M. miersiana E. Lucas 164 (K) Brazil JN661110 JN661060 JN660961 JN661011 JN660862 JN660912 M. myrcioides var. acrophylla O. Ribas 229 (ASU) Brazil JN661111 JN661061 JN660962 JN661012 JN660863 JN660913 M. myrcioides E. Lucas 503 (K) Brazil JN661113 JN661063 JN660964 JN661014 JN660865 JN660915 M. myrtoides M. Rossato 47 (MO) Brazil JN661117 JN661067 JN660968 JN661018 JN660869 JN660919 M. obtuse P. Brownless 1227 (CONC) Chile JN661114 JN661064 JN660965 JN661015 JN660866 JN660916 M. ovalifolia E. Lucas 259 (K) Chile JN661115 JN661065 JN660966 JN661016 JN660867 JN660917 M. ovata var. acutata F. Chagas 1979 (ASU) Brazil JN661116 JN661066 JN660967 JN661017 JN660868 JN660918 M. ovata var. nannophylla M. Mihoc 5162 (CONC) Brazil JN661118 JN661068 JN660969 JN661019 JN660870 JN660920 M. ovata var. ovata F. Gardner 19 (CONC) Chile JN661120 JN661070 JN660971 JN661021 JN660872 JN660922 M. ovata var. regnelliana J. Silva 18 (ASU) Brazil JN661119 JN661069 JN660970 JN661020 JN660871 JN660921 M. ovata var. regnelliana V. Souza 10621 (ASU) Chile JN661137 JN661087 JN660988 JN661037 JN660889 – M. oxysepala O. Ribas 2234 (ASU) Brazil JN661121 JN661071 JN660972 JN661022 JN660873 JN660923 M. parvifolia L. Landrum 5916 (CONC) Chile JN661122 JN661072 JN660973 JN661023 JN660874 JN660924 M. pilotantha E. Lucas 230 (K) Brazil JN661123 JN661073 JN660974 JN661024 JN660875 JN660925 M. pilotantha var. pilotantha C. Lohmann 35 (ASU) Brazil JN661124 JN661074 JN660975 JN661025 JN660876 JN660926 M. pinifolia F. Gardner 164 (CONC) Chile JN661125 JN661075 JN660976 JN661026 JN660877 JN660927 M. planipes C. Aedo 7378 (CONC) Chile JN661126 JN661076 JN660977 JN661027 JN660878 JN660928 M. reitzii E. Barbosa 945 (ASU) Brazil JN661135 JN661085 JN660986 JN661036 JN660887 JN660937 M. rufa S. Teillier 150795 (CONC) Chile JN661127 JN661077 JN660978 JN661028 JN660879 JN660929 M. rufescens E. Lucas 469 (K) Brazil JN661128 JN661078 JN660979 JN661029 JN660880 JN660930 M. schultzei E. Ruiz 8266 (CONC) Juan Fernández JN661136 JN661086 JN660987 – JN660888 JN660938 M. seriatoramosa J. Silva 2358 (MO) Brazil JN661130 JN661080 JN660981 JN661031 JN660882 JN660932 M. smithii R. García 533 (ASU) Brazil JN661129 JN661079 JN660980 JN661030 JN660881 JN660931 Myrcianthes cisplathensis L. Landrum 11233 (ASU) Uruguay JN661112 JN661062 JN660963 JN661013 JN660864 JN660914 Myrtus communis Botanical Garden Austria Austria JN661088 JN661038 JN660939 JN660989 JN660840 JN660890 Ugni molinae J. Murillo 4213 (CONC) Chile JN661131 JN661081 JN660982 JN661032 JN660883 JN660933 U. selkirkii F. Gardner 31 (CONC) Chile JN661132 JN661082 JN660983 JN661033 JN660884 JN660934 and Blepharocalyx eggersii for all regions nor M. schultzei Johow for (2008), who established pairwise interactions for each stem, where the trnK-matK region. pairing bases were coded as 1 and non-pairing bases as 0. In the Sequences were aligned with ClustalX (Thompson et al., 1997) ITS1 region the conserved angiosperm motifs GGCR-(4–7 n)-GYGY- and then corrected manually with Winclada (Nixon, 1999–2002) CAAGGAA (Liu and Schardl, 1994) were found. We also looked for according to the suggestions of Kelchner (2000). Gaps were coded the motifs GAATTGCAGAATCC, TTTGAAyGCA, CGATGAAGAACG- manually according to the simple indel coding method proposed TAGC (Harpke and Peterson, 2008; Jobes and Thien, 1997), and by Simmons and Ochoterena (2000). Total numbers of both indel the conserved EcoRV GATATC (Liston et al., 1996) in the 5.8S sites and indel events, and indel diversity were obtained with region. All ITS2 secondary structures showed four helices, helix DnaSP v.5.10.01 (Librado and Rozas, 2009). Alignments of ITS and III being the longest, a U–U mismatch in helix II, and an UGGU mo- ETS sequences were adjusted according to the secondary structure. tif near the apex of helix III (Schultz et al., 2005). The presence of all Folding predictions of secondary structures of the ITS1, 5.8S, these motifs suggests that all ITS sequences obtained in this study ITS2, and ETS were made at the mfold web server (http://mfold.r- are functional. ITS2 region was delimited in the Database http:// na.albany.edu/?q=mfold/RNA-Folding-Form)(Mathews et al., its2.bioapps.biozentrum.uni-wuerzburg.de/cgi-bin/index.pl?anno- 1999; Zucker, 2003). All parameters were used as default, with tator (Keller et al., 2009). and without input constraints. The secondary structure was chosen among those with the highest negative free energy value. Each 2.5. Phylogenetic analyses structure was compared with those ITS1 and ITS2 structures pro- posed by Biffin et al. (2007) for Myrtaceae. All sequences were 2.5.1. Maximum parsimony analyses (MP) aligned and corrected using secondary structure with 4Sale (Seibel Heuristic searches were performed using the parsimony algo- et al., 2006, 2008). Indels were coded following Tippery and Les rithm included in NONA (Goloboff, 1999), which uses Winclada J. Murillo-A. et al. / Molecular Phylogenetics and Evolution 62 (2012) 764–776 767
Table 2 List of primers.
Molecular Marker Pimer name DNA sequence Reference trnQ-50rps16 intergenic spacer trnQ(UUG) 50-GCGTGGCCAAGYGGTAAGGC-30 Shaw et al. (2007) MYtrnQR 50-AGTTGATGTAAAGGAAGATTTAGACTC-30 This study MYrps16F 50-GCGTAAAAWGAGGAAATGCTTAATG-30 This study rpS16x1 50-GTTGCTTTYTACCACATCGTTT-30 Shaw et al. (2007) rpl32-trnL intergenic spacer trnL(UAG) 50-CTGCTTCCTAAGAGCAGCGT-30 Shaw et al. (2007) rpL32-F 50-CAGTTCCAAAAAAACGTACTTC-30 Shaw et al. (2007) rpl16 intron rpl16-F71 50-GCTATGCTTAGTGTGTGACTCGTTG-30 Jordan et al. (1996) rpl16-R1516 50-CCCTTCATTCTTCCTCTATGTTG-30 Jordan et al. (1996) Partial matK gene + flanking trnK intron matK 700F 50-CAATCTTCTCACTTACGATCAACATC-30 Gruenstaeudl et al. (2009) matK1710R 50-GCTTGCATTTTTCATTGCACACG-30 Samuel et al. (2005) trnK-R3 50-CGG GGC TCG AAC CCG GA-3 Wicke and Quandt (2009) ITS AB101 50-ACGAATTCATGGTCCGGTGAAGTGTTCG-30 Sun et al. (1994) AB102 50-GAATTCCCCGGTTCGCTCGCCGTTAC-30 Sun et al. (1994) ITS-4 50-TCCTCCGCTTATTGATATGC-30 White et al. (1990) ETS + flanking 18S gene MyrtF 50-CTCCGTGCTGGTGCATCGAACTGC-30 Lucas et al. (2007) ETS-18S 50-GAGCCATTCGCAGTTTCACAG-30 Wright et al. (2001)
as interface (Nixon, 1999–2002). Parsimony ratchet analyses were generated in each run were evaluated in Tracer v1.4 (Rambaut performed with the following strategy: 10 replicates, 1000 itera- and Drummond, 2007) to determinate effective sample size, con- tions replicate, holding 10 trees per iteration, sampling 10% of vergence of both runs, mixing of the MCMC chains, and Burn-in val- the characters, and 10 random constraint levels. The characters ues for eliminating sample trees found before the stationary phase. were assessed as unordered and equally weighted (Fitch, 1971). From the remaining trees a majority rule consensus tree was recon- Unsupported branches were collapsed and polytomies were al- structed and posterior probabilities (PPs) calculated. lowed. Multiple searches were carried out until no shorter equally parsimonious trees were obtained. Branch supports were assessed 2.5.3. Network analysis using parsimony bootstrapping with 1000 replicates, 10 random To evaluate conflicting phylogenetic signals, concatenated data search replications each, one starting tree per replicate, 100 max- sets of all molecular markers were evaluated with Neighbor Net trees, and Tree Bisection and Reconnection (TBR) on. Several inde- analysis, using uncorrected p-distances as implemented in Splits- pendent analyses were performed. Each region was evaluated Tree4 version 4.11.3 (Huson and Bryant, 2006). Parsimony uninfor- separately, and then several combined data sets were also evalu- mative sites were excluded. Bootstrap support for splits was ated, including all chloroplast markers (cpDNA), all nuclear regions calculated using 1000 replicates. (nDNA), cpDNA + ETS, cpDNA + ITS, all combined data (cpnDNA), and cpnDNA + gaps. Congruence among each combined data set 2.6. Statistical analysis was determined by using the Partition homogeneity test (ILD test) (Farris et al., 1994) implemented in PAUP⁄v4.10 (Swofford, 2002). Saturation of substitutions was evaluated with the Xia index For each ILD test 1000 replicates were carried out, each with 10 using DAMBE v 5.2.13 (Xia, 2001; Xia and Xie, 2001). The diversity random addition sequence replicates, holding 10 trees per repli- of nucleotides for each marker and the rate of divergence for each cate, TBR branch swapping, and MulTrees on. clade were estimated by Mega 4 (Tamura et al., 2007) following phylogenetic results recovered from all combined marker analyses 2.5.2. Bayesian analyses (BA) (Fig. 3) and among groups of species ordained by geographic distri- Bayesian inference of phylogenetic reconstruction (Huelsenbeck bution in clades I–III from Chile and clade IV from Brazil. and Ronquist, 2001) was performed using MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003), with Markov Chain Monte Carlo (MCMC) 3. Results providing the heuristic search for a maximum likelihood model of nucleotide substitution. To select the best model of base substitu- 3.1. Chloroplast markers tion of DNA, each individual marker data set was evaluated accord- ing to the Akaike information criterion using Modeltest 3.7 (Posada All chloroplast markers showed low variability. The trnQ- and Crandall, 1998) under MrMTgui Interface 1.0 (Nuin, 2007). The 50rps16 spacer displayed more variables (7.44%) and more best models were TVM + G (trnQ-50rps16, rpl32-trnL), K81uf + I + G parsimony informative characters (4.36%); all other regions are (rpl16), GTR + G (trnK-matK), TIM + G (ETS, ITS1), JC + I (5.8S), less variable, with only 2.95–3.72% parsimony informative charac- TVM + I + G (ITS2). Each region was evaluated separately and in ters (Table 3). this is also shown with the nucleotide diversity combination, in this case a partitioning model was used, allowing value, the trnQ-50rps16 region revealing 1.37–1.5 more diversity for the best substitution model for each marker. Two independent than other chloroplast markers (Table 4). All chloroplast sequences runs integrated each analysis, each with four chains. Analyses were were not significantly saturated (Table 5). Plastid markers have run between two and ten million generations until the average high content of T/A bases (Table 4). Six poly T/A regions are present standard deviation of split frequencies became less than 0.01. For in the rpl32-trnL region with a length from 7 to 12 bp, whereas the each chain one tree was saved every 10 generations. All trees rpl16 intron has two poly T/A regions from 6 to 7 to 12 bp. The 768 J. Murillo-A. et al. / Molecular Phylogenetics and Evolution 62 (2012) 764–776
Table 3 Summary of MP analyses of plastid and nuclear markers.
trnQ- rpl32- rpl16 trnK- cpnDNA cpDNA + gaps ETS ITS nDNA cpDNA + ETS cpDNA + ITS cpDNA cpDNA + ETS + ITS + gaps 50rps16 trnL matK Number of 50 50 50 49 50 50 50 49 50 50 50 50 50 taxa Character 1465 885 1005 1083 4438 4628 502 642 1145 4940 5080 5582 6226 Constant 1292 795 904 978 3969 379 516 895 4348 4485 4865 characters (88.19) (89.83) (89.95) (90.34) (89.43) (75.49) (80.37) (%) Variable 109 57 65 73 304 68 61 (9.5) 129 372 365 433 characters (7.44) (6.44) (6.46) (6.74) (6.84) (13.54) (%) NP-informative 64 (4.36) 33 36 32 165 263 (5.68) 55 65 121 220 230 285 419 characters (3.72) (3.58) (2.95) (3.71) (10.95) (10.12) (%) No. of most 12 135 98 188 12 290 81 140 794 440 185 8 1192 parsimonious trees Length 95 51 62 46 272 574 202 160 325 419 499 641 1050 Consistency 0.77 0.66 0.62 0.76 0.66 0.48 0.66 0.51 0.48 0.59 0.54 0.52 0.44 index CI Retention 0.92 0.81 0.87 0.9 0.86 0.73 0.61 0.68 0.63 0.79 0.76 0.73 0.66 index (RI) trnK-matK region has the least number of indels (22 sites, nine events, indel diversity 0.855) (Table 6). Regions with both the 0 Table 5 greatest indel diversity and indel length are trnQ-5 rps16 (9.418, Substitution saturation. 261) and rpl32-trnL (8.797, 156). Due to low DNA quality several sequences were partially amplified, e.g., most trnK-matK sequences Iss Iss.c p only amplified between 758 and 1031 bp. B. salicifolius did not am- trnQ-50rps16 0.011 0.666 <0.0001 plify 521 bp for the rpl32-trnL region and M. ovata var. regnelliana rpl32-trnL 0.071 0.678 <0.0001 rpl16 0.047 0.718 <0.0001 only amplified 673 bp for trnQ-50rps16 region. trnK-matK 0.031 0.717 <0.0001 MP analyses from individual chloroplast regions gave strict con- ETS 0.091 0.681 <0.0001 sensus trees with low resolution (data not shown). The rpl32-trnL ITS 0.041 0.709 <0.0001 region displayed the lowest resolution (data not shown), whereas Iss = index of substitution saturation. 0 trnQ-5 rps16 region had the most resolved tree (Fig. 1A). However, Iss.c = critical value. all topologies are nearly congruent and show Myrceugenia to be monophyletic (Myrceugenia clade), except for M. fernandeziana appears in different placement within the outgroups. Brazilian spe- Myrceugenia exsucca andMyrceugenia lanceolata, is highly supported cies appear as a derived monophyletic group. Furthermore, there is (BS = 99%, PP = 1.0) by six non-homoplasious substitutions. This no evidence of a monophyletic origin of the ‘‘Myrceugenia group’’ clade was also recovered from the trnQ-50rps16, rpl32-trnL, rpl16 (sensu Lucas et al., 2007). All of these relationships are completely and trnK-matK analyses (Fig. 1). The next clade (clade II), consisting congruent with results rising from Bayesian analysis (BA) (Fig. 1), of Myrceugenia leptospermoides, Myrceugenia planipes, Myrceugenia but with more resolution. parvifolia and Myrceugenia pinifolia received high support (BS = 97, There was no significant incongruence among the four-chloro- PP = 0.97) and is supported by one non-homoplasious substitution. plast regions (P = 0.57). Therefore, we combined all markers into a This clade was only recovered from the trnQ-50rps16 spacer single data set with a total length of 4438 sites. Both MP and BA (Fig. 1A). The third clade (clade III), comprising the remaining Chil- analyses of the combined data set agree with separate analyses of ean species, was only recovered from BA for all chloroplast markers, individual molecular markers (Fig. 2A). M. fernandeziana appears but only poorly supported (PP = 0.64%) by ten substitutions, six of in a clade with B. salicifolius (BS = 97%, PP = 0.69), whereas most these without homoplasy. In the last clade (clade IV) all the Brazilian Myrceugenia species are included within a monophyletic clade species appear; this clade is highly supported (BS = 99, PP = 1.0), but (BS = 100%, PP = 1.0), with 13 synapomorphic substitutions, of with low resolution. Average evolutionary divergences over se- which nine are non-homoplasious. Chilean species are recovered quence pairs within clades revealed that Chilean clades (I, II, III) con- in three clades: the earliest diverging clade (clade I), comprising tain more divergence than the Brazilian clade (IV), except for rpl16 Myrceugenia obtusa, Myrceugenia correifolia, Myrceugenia rufa, that is lower (Table 7).
Table 4 Nucleotide compositions of plastid and nuclear regions.
Marker Alignment sites (range) bp A T G C GC% Nucleotide diversity trnQ-50rps16 1483 (1206–1384) 0.3795 0.3516 0.1353 0.1335 0.2631–0.3025 0.01393 rpl32-trnL 885 (795–840) 0.32466 0.39901 0.13816 0.13817 0.2825–0.3203 0.01015 rpl16 1005 (817–967) 0.27693 0.41009 0.14939 0.16359 0.3048–0.3276 0.00995 trnK-matK 1083 (1056–1064) 0.34544 0.32224 0.15217 0.18015 0.3308–0.3919 0.00928 ETS 502 (455–470) 0.18842 0.31794 0.25902 0.23461 0.4792–0.5185 0.02671 ITS 642 (602–616) 0.21896 0.21436 0.27892 0.28777 0.5548–0.5873 0.02613 ITS1 256 (239–245) 5.8S 160 (153–160) ITS2 226 (208–217) J. Murillo-A. et al. / Molecular Phylogenetics and Evolution 62 (2012) 764–776 769
Table 6 Indel characteristics of plastid and nuclear regions.
trnQ-50rps16 rpl32-trnL rpl16 trnK-matK ETS ITS Range of indel length 1–261 1–156 1–29 1–7 1–10 1–6 Total number of indel sites 374 391 208 22 73 78 Total number of indel events 71 52 32 9 60 62 Indel diversity k(i) 9.418 8.797 4.73 0.855 5.433 6.648
Fig. 1. Majority consensus tree of species of Myrceugenia and outgroups from Bayesian analyses of (A) trnQ-5’rps16, (B) rpl16, and (C) trnK-matK regions. Numbers above branches are Bayesian posterior probabilities; numbers below branches are bootstrap percentages. Branches are colored according to their main geographical distribution: red, Brazilian species; blue, Chilean species; green, M. fernandeziana. The scale indicates substitutions per site.
3.2. Nuclear markers Table 4), average evolutionary divergence over sequence pairs (Table 7), and lower indel variability (Table 6) than the plastid Nuclear markers displayed more variability (10.12–10.95%, markers. ETS and ITS sequences did not show significant saturation Table 3), diversity (1.9–2.9-fold, Table 4), GC content (1.2–2.2-fold, of nucleotide substitution (Table 5). Both the strict consensus trees 770 J. Murillo-A. et al. / Molecular Phylogenetics and Evolution 62 (2012) 764–776
Fig. 2. Bayesian trees of species of Myrceugenia and outgroups resulting from combined analyses of (A) cpDNA and (B) nDNA sequences. Numbers above branches are Bayesian posterior probabilities; numbers below branches are bootstrap percentages. Brach red, Brazilian species; green, blue and yellow, Chilean species, and violet, M. fernandeziana. The scale indicates substitutions per site. J. Murillo-A. et al. / Molecular Phylogenetics and Evolution 62 (2012) 764–776 771
Table 7 Estimates of average evolutionary divergence over sequence pairs within Chilean and Brazilian clades (average/standard deviation).
Group trnQ-50rps16 rpl32-trnL rpl16 trnK-matK ETS ITS Outgroups 0.02010/0.00215 0.01236/0.00210 0.01449/0.00244 0.01654/0.00235 0.05130/0.00465 0.04613/0.00630 Chile Clade I–III 0.00717/0.00133 0.00901/0.00220 0.00346/0.00111 0.00397/0.00120 0.01783/0.00208 0.02069/0.00350 Clade I 0.00191/0.00079 0.00426/0.00157 0.00250/0.00108 0.00219/0.00094 0.01802/0.00281 0.02907/0.00604 Clade II 0.00359/0.00118 0.00510/0.00180 0.00219/0.00115 0.00503/0.00200 0.01809/0.00308 0.01382/0.00376 Clade III 0.00714/0.00150 0.00496/0.00161 0.00220/0.00104 0.00205/0.00119 0.01582/0.00297 0.01622/0.00409 Brazil Clade IV 0.00313/0.000066 0.00278/0.00069 0.00474/0.00097 0.00275/0.00081 0.01075/0.00142 0.01243/0.00207
from MP and majority rule consensus tree from BA obtained from low resolution, but three highly supported (BS > 0.86) subclades individual markers showed poor resolution (data not shown). Con- were recovered. sensus trees from combined nuclear markers are partially congru- ent with those from plastid markers (Fig. 2B). Myrceugenia appears 3.3. Congruence monophyletic, with most species of the genus, except for M. fernandeziana, residing in a highly supported (BS = 96% PP = 1.0) The partition homogeneity test for nDNA, cpDNA + ETS, group held together by seven synapomorphic substitutions, three cpDNA + ITS, and cpnDNA show character incongruence (P = 0.01). of these non-homoplasious. M. rufa is sister to the rest of the spe- All analyses show congruence among major clades, but within cies of Myrceugenia; this relationship only emerges in BA analyses clades poor resolution emerges, which could explain lack of congru- from ITS and from combined nuclear marker analyses. The Chilean ence reported by the ILD test. All topologies displayed short species are poorly resolved, whereas Brazilian species appear in a branches, mostly poorly supported, especially among Brazilian spe- highly supported (BS = 93% PP = 0.94) monophyletic group held cies. Such conditions have been interpreted by Wendel and Doyle together by four homoplasious synapomorphies. A clade consisting (1998) as a soft incongruence, which might disappear with addi- of Myrceugenia alpigena and Myrceugenia rufescens (BS = 91% tional data. Gatesy et al. (1999) demonstrated that concatenating PP = 0.94) is sister to the rest of the Brazilian species. This relation- of truly incongruent data sets could still increase resolution and ship is not found in all other analyses. The most derived clade has branch support. Furthermore, there is no evidence of the presence
Fig. 3. Bayesian tree resulting from all sequence data (cpnDNA). Numbers above branches are Bayesian posterior probabilities; numbers below branches are bootstrap percentages. Branches are colored according to Fig. 2. The scale indicates substitutions per site. 772 J. Murillo-A. et al. / Molecular Phylogenetics and Evolution 62 (2012) 764–776 of pseudogenes in nuclear regions that could explain the incongru- secondary structure: a bulge arising from a mismatch at the middle ence. Therefore we combined all genomic markers. of Helix II, and other mismatch whereby the Myrceugenia clade has the Helix VI shorter than the outgroups or M. fernandeziana. Clade I 3.4. Combined analysis is supported by a large deletion of 168 bp, occurring between posi- tions 342 and 510 in the spacer trnQ-50rps16. Clades II, III and IV MP and BA analyses from cpDNA + ETS, cpDNA + ITS and have a synapomorphic deletion of 28 bp (positions 244–272) of cpnDNA showed similar consensus trees, except that the strict con- the rpl16 intron. Clade IVa has the insertion ATTAC in the trnQ- sensus trees of MP recovered from cpDNA + ETS and cpDNA + ITS 50rps16 region. A deletion of 11 bp (position 290–300) in rpl32-trnL show least resolution within clade IV; all of these, however, are is a synapomorphy for Ugni. nearly congruent with those from combined chloroplast markers. The BA consensus tree from cpnDNA (Fig. 3) shows that the Myr- 4. Discussion ceugenia clade is strongly supported, as well as clades I to IV. Clade IV appears with two subclades (Clade IVa, IVb); these emerge from 4.1. Polyphyly of Myrceugenia all combined partitions and are quite similar to those from cpDNA (Fig. 2A) and trnQ-50rps16 (Fig. 1A) analyses. These subclades from All analyses strongly indicate that Myrceugenia is monophyletic BA have relatively high support, but in MP support is low. only when the Juan Fernández species, M. fernandeziana, which Neighbor Net analysis. Splits graph analysis reveals that species shows no relationship with other species of the genus, is excluded. of Myrceugenia are distributed in four splits (Fig. 4), but M. fernan- The clade containing the remaining species of Myrceugenia is sup- deziana groups with outgroups taxa as also shown in the phyloge- ported by 12 substitutions (three nuclear and nine plastid), an netic analyses. All of these splits are strongly compatible with insertion of trnK-matK, two gaps from rpl32-trnL, and two struc- topologies recovered from MP and BA analysis, except that M. ovata tural differences within ETS secondary structure. None of these var. ovata and M. ovata var. nannophylla cluster within split II. Sup- traits are present in M. fernandeziana nor in other genera of tribe port between split II and III is low, but other splits are strongly sup- Myrteae examined. This strongly suggests that M. fernandeziana ported. Conflicting phylogenetic signals occur within split I and does not belong to Myrceugenia. also within split IV. M. fernandeziana was included in the genus Nothomyrcia by Kausel (1948), which was considered by McVaugh (1968) to have 3.5. Indel information unclear position within tribe Myrteae, and related in some fashion to the ancestor of the tribe. Landrum (1981b) considered it as a Similar topologies are recovered from analyses with or without early diverging species within Myrceugenia, closely related to two gaps. Inclusion of coded indels reduced bootstrap support, CI, RI, Brazilian species, Myrceugenia campestris and M. rufescens, sup- and resolution. Analyses including all markers and coded indels ported by features of the indumentum, inflorescence, and flowers. reveal the Myrceugenia clade to have three non-homoplasious indel According to results in the present study, these morphological sim- synapomorphies: an insertion of TAAA for the trnK-matK region, ilarities appear to be convergences, possibly favored by similar and two gaps, AAGTGATGA and TTMAAAKT, for the rpl32-trnL climatic conditions in which all these species occur, i.e., a foggy region. In Addition, two indel synapomorphies exist in the ETS subtropical forests (Landrum, 1981a). Morphological differences
Fig. 4. Split graph resulting from Neighbor Net analysis using concatenated data sets of all molecular markers. Numbers are Bootstrap values. Splits are colored according to Fig. 2. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) J. Murillo-A. et al. / Molecular Phylogenetics and Evolution 62 (2012) 764–776 773 to the remaining Myrceugenia species are scarious bracteoles, (Landrum, 1981a). All results are consistent, in suggesting that which are caducous at anthesis, and a leaf midvein that is not im- these species are sister to the remaining members of the genus. pressed, whereas in the rest of the species the bracteoles are rarely Clade II is recovered from all combined analyses (Fig. 3) and also scarious and usually persistent, and the midvein of the leaves is from the trnQ-50rps16 analysis (Fig. 1A) with strong support, but in impressed (Landrum, 1981a). Preliminary results regarding taxo- MP analyses alone, it has poor support. Neighbor Net analysis con- nomic position of this species, including 30 genera of tribe Myrteae firms a relationship among these species, but in this split Chilean (Murillo, 2011), suggest that M. fernandeziana is an independent varieties of M. ovata are also included (Fig. 4). Such a relationship lineage related to Blepharocalyx. These results support both what does not appear in any other phylogenetic analysis, which falls Kausel (1948) considered as an independent genus, and McVaugh poorly supported within clade III from combined phylogenetic (1968) considered as an independent, relatively unsuccessful analyses. These varieties are recovered as an independent clade lineage. from the spacer trnQ-50rps16 analysis. The other individual chloro- plast markers reveal no resolution, whereas nuclear markers show 4.2. Phylogenetic relationships within Myrceugenia them to be biphyletic. Despite the fact that a monophyletic origin of the other Brazilian varieties was not seen, Landrum (1981a) still Myrceugenia splits into four clades (Fig 3). The Chilean species kept them under the same species because he could not find mor- are distributed into three clades, whereas all Brazilian species are phological characters to separate them. All molecular analyses, included in a derived monophyletic group. All of these relation- however, strongly support their separation. ships are supported by Neighbor Net analysis (Fig. 4), which dis- Relationships among M. chrysocarpa, M. colchaguensis and M. plays a split graph strongly congruent with the combined schultzei recovered from cpDNA, cpDNA + ETS, cpDNA + ITS and analysis. These results affirm concatenation of both chloroplast trnQ-50rps16 analyses all are strongly supported. This is confirmed and nuclear markers, which were shown to be incongruent by with Neighbor Net analysis, but this and MP analyses show low the ILD test, but which improved topology resolution as suggested support for relationships between M. chrysocarpa and the other by Gatesy et al. (1999). Incongruence has also been found when two species. Results from BA of trnQ-50rps16 spacer are highly con- there are pseudogenes in the nuclear regions, but no evidence of gruent with those from combined analysis, showing its high phylo- these non-functional regions has been found in any of the genetic signal. The tree from this analysis (Fig. 1A) shows that sequences analyzed. Neighbor Net analysis shows incongruence these three species are sister to those in Brazil. This relationship in splits I and IV, which could be due to recombination, hybridiza- was partially found in the phylogenetic analysis by Landrum tion, gene conversion, gene transfer, or sampling error (Bryant and (1981a), whereby M. chrysocarpa appears as the earliest diverging Moulton, 2004). According to the phi test, however, there was no species of a clade formed by M. colchaguensis and 22 Brazilian taxa. statistically significant evidence for recombination (p = 0.239). This This would suggest a common ancestry for these Chilean and might suggest therefore that incongruencies are due to low levels Brazilian species. M. schultzei was considered by Landrum of sequence variability, which can obviously cause lack of true phy- (1981b) to be an ancient splitting taxon within Myrceugenia and logenetic signal (Wendel and Doyle, 1998). closely allied to M. lanceolata and M. exsucca. In our study, how- Landrum (1981b), based on morphological data, proposed that ever, none of these relationships are observed; M. schultzei in fact, Myrceugenia could be split into five groups, each one including spe- appears to be one of the most derived species within the Chilean cies from both Chile and Brazil. A clade recovered from ITS analysis group. consisting of the three varieties of M. alpigena, M. rufescens from The Brazilian clade is strongly supported (BS = 100% PP = 1.0), Brazil, and the Chilean M. schultzei and Myrceugenia colchaguensis, also consistent with Neighbor Net analysis (BS = 100%), but with and another clade emerging from ETS analyses that includes Myr- poor resolution. This lack of resolution has been interpreted as ceugenia cucullata and M. rufescens from Brazil and M. parvifolia and resulting from saturation of variable sites, such that phylogenetic M. correifolia from Chile, could justify those relationships. These signal is not recovered (von Dohlen and Moran, 2000). There is clades, however, are poorly supported (0.69 PP, 0.7 PP, respec- no significant evidence, however, for saturation in either chloro- tively) and they are not recovered from either chloroplast markers plast or nuclear sequences (Table 5). We interpret these ambiguous or any partitioned data set analyses. Relationships recovered by relationships as being due to low number of informative characters Landrum (1981b) show only seven Brazilian species appearing (5.1% combined data) and lack of synapomorphic characters as close to Chilean species, whereas the others remain in a separate shown by CI = 0.52 and RI = 0.73 in the parsimony analysis. The clade. According to our analyses, we might infer that lack of sepa- average evolutionary divergence over sequence pairs demonstrates ration of these two lineages in the Landrum analyses might be due that Chilean species show between1.44- and 3.24-fold higher to paucity of informative characters. It is not easy to establish com- divergence than Brazilian species (Table 7), except for rpl16 that parisons between molecular and morphological data for each is 1.36-fold lower, suggesting independent evolutionary histories clade; because there seems to be no correlation between molecular for these disjunct groups. The low resolution, short branches, and data and morphological data; it will be necessary, therefore, to look high species diversity may be correlated with rapid radiation for new morphological and anatomical data to help clarify relation- (Schwarzbach and Kadereit, 1995; Baldwin and Sanderson, 1998; ships found in this study. Fishbein et al., 2001; Hughes and Eastwood, 2006; Miwa et al., Clade I, composed of M. lanceolata, M. exsucca, M. rufa, M. correi- 2009) as emphasized by both lack of phylogenetic signal among folia, and M. obtusa, was recovered from all analyses, except from species of split IV and low resolution within the diverse clade IV. that with nuclear markers. Little internal resolution exists, but Rapid radiation could help explain the lack of definitive molecular BA analyses from ITS and from combined ITS + ETS analyses show and morphological characters (von Dohlen and Moran, 2000), at M. rufa to be the earliest diverging species. However, the incongru- least in the Brazilian species. ent position of this species or any other species, when they are compared with the topology found with chloroplast markers, 4.3. Relationship within the ‘‘Myrceugenia group’’ may be due to lack of resolution among species (Fig. 2B). Resolu- tion increased, however, especially among Chilean species, when The ‘‘Myrceugenia group’’ was proposed by Lucas et al. (2007) all data were combined (Fig. 3), which is a general result suggested to include Blepharocalyx, Luma, and Myrceugenia. All analyses in by Wendel and Doyle (1998). The three first-listed species were this study, however, show no evidence of monophyly for this treated as being closely related from the morphological analyses group. Depending on data analyzed, genera of this complex appear 774 J. Murillo-A. et al. / Molecular Phylogenetics and Evolution 62 (2012) 764–776 in different positions associated with other genera. Species belong- (Meudt and Simpson, 2006), which could suggest a local dispersal ing to Luma form the only consistently monophyletic clade. Species pattern for development of the vegetation (Arroyo pers. comm.). of Blepharocalyx appear biphyletic as was displayed by Lucas et al. During the Pliocene, the clade III shows a dispersal to the Juan Fer- (2007); evidence for their monophyly was only recovered from ITS nandez Archipelago now represented by M. schultzei from Masafu- Bayesian analysis with high support (PP = 0.91). era. The analysis suggests Brazilian Myrceugenia diverged in the According to Landrum (1981b) these three genera and Myrcian- Lower Miocene from ancestors in southern Chile, but its diversifi- thes belong to separate lineages having arisen from ancestors of cation in southeastern Brazil began during Middle Miocene, in a each of the three subtribes recognized by Berg (1855–1856), process of colonization from South to North. whereas Luma would have arisen from the tribe ancestor. All of Separation of southern from northern South America by the these genera share anatomical and floral features (e.g., some ves- Paranaense Sea (15–13 Ma) (Hernández et al., 2005), plus the sels with scalariform perforation; simple, uniflorous, dichasial or final early Pliocene uplift of the Andes and the ‘‘Pampean bracteate shoot inflorescences; tetramerous flowers; free calyx- Mountain Range’’ in central Argentina (Pascual et al., 1996), all lobes; 2–4 locular ovaries, numerous ovules; free cotyledons; and resulted in completion of the arid diagonal. This began to devel- membranous testa), which would be regarded as plesiomorphies. op at the end of the Miocene (Hinojosa and Villagrán, 1997; Lucas et al. (2007) proposed that these characteristics are, in fact, Gregory-Wodzicki, 2000), which could have affected distribu- synapomorphies of the ‘‘Myrceugenia group’’, but in their study tions of both arthropods (Donato, 2006; Roig-Juñent et al., this group had low support and only appears when data are com- 2006) and plants (Ritz et al., 2007; Marquínez et al., 2009). It bined. Analyses presented here do not establish whether all of is likely, therefore, that the same events occurring during the these genera represent a very old linage arising from ancestral Miocene caused Myrceugenia to remain confined to Central Chile groups of tribe Myrteae, but what is possible to propose is that and southeastern Brazil, regions of similar climatic conditions all these genera do not belong to a monophyletic group. (Landrum, 1981b). Molecular calibration studies for the flora of South America are 4.4. Biogeographical implications scarce. Estimated age for divergence of the Chilean species of Myr- ceugenia (Murillo, 2011) and Drimys (Marquínez et al., 2009) from Myrceugenia is a genus that has originated in southern South those present in Brazil, is between 16 and 13 Ma. This age estima- America (Landrum, 1981b; Lucas et al., 2007), with two centers tion coincides with the formation of the Paranaense Sea of species diversity in Central Chile and southeastern Brazil. This (Hernández et al., 2005). Disjunct distribution patterns in many disjunct distribution is also seen in other taxa such as Alstroemeria, South American genera (Villagran and Hinojosa, 1997; Landrum, Araucaria, Azara, Escallonia, and Weinmannia (Landrum, 1981b). 1981b), which also match ages of divergence of Myrceugenia and Distribution of the South American flora has been explained by Drimys would support the hypothesis that the Paranaense Sea climatic and geological changes occurring during the Neogene could have acted as cause of vicariance. This would have had great (Landrum, 1981b; Villagran and Hinojosa, 1997). importance for the distribution and diversity of the flora and fauna The earliest fossil records of Myrtaceae in the southern South of South America (Webb, 1995). In Australia, there is a very rich America are pollen from the Campanian (83.5–70.6 Ma) (Poole endemic flora distributed into two zones separated by a wide arid and Cantrill, 2006; Prámparo et al., 2007). Remains of leaves and zone where many taxa occurred disjunctly. This is similar to the wood from the Late Cretaceous have been found that have been pattern in southern South America. Divergences among those lin- assigned to Eugenia, Luma (Poole et al., 2001, 2003) and Myrcia eages in Australia have also been interpreted to be due to an (Barreda and Palazzesi, 2007). The most ancient fossil of Myrceuge- ancient vicariant event (Crisp and Cook, 2007). nia is M. chubutense, described by Ragonese (1980) from wood remains in southwestern Argentina (Province Chubut) from early Acknowledgments Paleocene (61.7–65.5 Ma) deposits. Other fossils assigned to this genus have been reported from leaves from central Argentina The authors thank CONYCIT (24090098) of Chile and Univer- (early Eocene, 47–52 Ma; González et al., 2003; Wilf et al., 2005) sity of Concepción (DIUC 209.111.054-1.0) for financial support. and central Chile (early Eocene; Gayo et al., 2005), and early Herbaria ASU, CONC, K, MO and RB sent us material for DNA Miocene (21 Ma; Villagrán and Hinojosa, 2005). Fossil records, extraction. Eva Lucas from Royal Botanic Gardens, Kew, and therefore suggest that Myrceugenia is a very old lineage, as was in- Edith Karpino from Jodrell Laboratory, Kew sent us DNA aliquots ferred by McVaugh (1968) and Landrum (1981b), having been of some species. Herbarium VEZ sent specimens of Blepharocalyx widely distributed in southern South America (Landrum, 1981b), eggerssi for morphological study. Jim Solomon, Missouri Botanical from early Paleocene. Garden, Rafaela Forzza, Rio de Janeiro Botanical Garden, Frank Murillo (2011) selected to M. chubutense and Myrceugenelloxylon Schumacher, Botanical Garden, University of Vienna, all assisted antarcticus, to perform a calibration analysis for the tribe Myrteae. us in providing material. Labwork at Concepción was facilitated The latter is a fossil from the Upper Cretaceous (Maastrichtian, by Patricia Gómez, Mariela González and Angela Carrasco, and 68.5–65 Ma, Poole et al., 2003) to the Middle Eocene of Antarctica in the Molecular Systematics Laboratory Department of System- (49–43 Ma, Poole and Cantrill, 2006) that shows wood anatomy atic and Evolutionary Botany, University of Vienna, by Rosabelle similar to moderm L. apiculata. These results suggest that Myrteae Samuel, Elfriede Grasserbauer, Gudrun Kohl, Verena Klejna, Patri- is older than what has been proposed by Sytsma et al. (2004) and cio López, and Walter Till. We thank two anonymous reviewers Biffin et al. (2010) based on fruits remains of Paleomyrtinaea princ- for their comments and suggestions on the manuscript. JM was etonensis, a fossil described from the Palaeocene (56 Ma) of North supported by a fellowship from the University of Concepcion- America (Pigg et al., 1993), which would agree with the extensive MECESUP (UCO0708) and from CONICYT for a stay at Vienna and old fossil record for the family in southern South America. University. Partially supported by FWF Grant No. P21723-B16 Biogeographic analyses (Murillo, 2011) of taxa in clade I suggest to TFS. that species dispersed to northern Chile between the Lower and Middle Miocene. 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